Gene cloning, optimized expression and immunogenicity evaluation of tetanus toxin fragment C / 南方医科大学学报
Journal of Southern Medical University
;
(12): 731-735, 2008.
Artículo
en Chino
| WPRIM
| ID: wpr-280108
ABSTRACT
<p><b>OBJECTIVE</b>To obtain highly purified tetanus toxin fragment C (TTC) with good immunogenicity.</p><p><b>METHODS</b>The gene fragment encoding TTC was amplified from Clostridium tetani plasmid DNA by PCR, inserted into the vector pET43.1a (+) and expressed in E. coli BL21(DE3)plysS. After purification using Ni2+-chelate affinity chromatography, the expressed fusion protein was digested by thrombin and the resultant TTC protein was purified with Ni2+-chelate affinity chromatography followed by identification with SDS-PAGE and Western blotting. The purifed TTC protein was then used to immunize mice to test its immunogenecity.</p><p><b>RESULTS</b>The 1373-bp gene fragment encoding TTC was obtained, and the constructed recombinant expression vector pET43.1a (+)-TTC was successfully expressed in E. coli BL21(DE3)plysS. SDS-PAGE identified a recombinant fusion protein with relative molecular mass (Mr) of 117 000, which accounted for 22% of the total bacterial protein. The TTC protein with Mr of 50 000 was obtained after purification of the thrombin digestion products of the fusion protein, with a purity reaching 95.5%. Both the fusion protein and TTC protein could be recognized by anti-tetanus toxin antibody as shown by Western blotting. The titer of the anti-serum from mice immunized with the TTC protein was 125 600, and the anti-serum could specifically bind to tetanus toxin.</p><p><b>CONCLUSION</b>Highly purified and immunogenetic TTC protein has been successfully obtained, which provides a good model antigen for studying antigen presentation and immune responses in vivo.</p>
Texto completo:
Disponible
Índice:
WPRIM (Pacífico Occidental)
Asunto principal:
Fragmentos de Péptidos
/
Toxina Tetánica
/
Proteínas Recombinantes de Fusión
/
Toxoide Tetánico
/
Clonación Molecular
/
Electroforesis en Gel de Poliacrilamida
/
Alergia e Inmunología
/
Escherichia coli
/
Vectores Genéticos
/
Genética
Idioma:
Chino
Revista:
Journal of Southern Medical University
Año:
2008
Tipo del documento:
Artículo
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